Phylogenetic analysis and functional characterization of norcoclaurine synthase involved in benzylisoquinoline alkaloids biosynthesis in Stephania tetrandra.

Benzylisoquinoline alkaloids (BIAs) are a class of secondary metabolites that possess diverse pharmaceutical properties and are exclusively accumulated in specific plant genera. The Pictet-Spengler condensation, catalyzed by norcoclaurine synthase (NCS), represents a key enzymatic reaction in the biosynthetic pathway of BIAs. While NCS genes have been identified in several plant families such as Papaveraceae, Berberidaceae, and Ranunculaceae, no NCS genes have been reported in Menispermaceae, which is another genus known to accumulate BIAs. Here, NCSs were isolated and functionally characterized from the Menispermaceae family plant Stephania tetrandra. In vitro enzyme assay identified two functional StNCSs which could catalyze the formation of (S)-norcoclaurine. These functionally characterized genes were then integrated into engineered yeast to enable the production of norcoclaurine. Phylogenetic analysis of the NCS enzymes revealed that the StNCSs predominantly clustered into two clades. The functional StNCSs clustered with known NCSs, highlighting the presence of a specific NCS catalytic domain. This study not only provides additional genetic components for the synthetic biology-based production of BIAs in yeast but also contributes to the understanding of the phylogenetic relationships and structure-function relationship of NCS genes involved in the origin and production of BIAs.

Structure-function analysis of CYP719As involved in methylenedioxy bridge-formation in the biosynthesis of benzylisoquinoline alkaloids and its de novo production.

Benzylisoquinoline alkaloids (BIAs) are a type of secondary metabolite with clinical application value. (S)-stylopine is a special BIA which contains methylenedioxy bridge structures. CYP719As could catalyze the methylenedioxy bridge-formation on the A or D rings of protoberberine alkaloids, while displaying significant substrate regiospecificity. To explore the substrate preference of CYP719As, we cloned and identified five CyCYP719A candidates from Corydalis yanhusuo. Two CyCYP719As (CyCYP719A39 and CyCYP719A42) with high catalytic efficiency for the methylenedioxy bridge-formation on the D or A rings were characterized, respectively. The residues (Leu 294 for CyCYP719A42 and Asp 289 for CyCYP719A39) were identified as the key to controlling the regioselectivity of CYP719As affecting the methylenedioxy bridge-formation on the A or D rings by homology modeling and mutation analysis. Furthermore, for de novo production of BIAs, CyCYP719A39, CyCYP719A42, and their mutants were introduced into the (S)-scoulerine-producing yeast to produce 32 mg/L (S)-stylopine. These results lay a foundation for understanding the structure-function relationship of CYP719A-mediated methylenedioxy bridge-formation and provide yeast strains for the BIAs production by synthetic biology.

[Modification of C20 oxidase in tanshinone biosynthesis pathway].

Tanshinones are one of the main effective components of Salvia miltiorrhiza, which play important roles in the treatment of cardiovascular diseases. Microbial heterogony production of tanshinones can provide a large number of raw materials for the production of traditional Chinese medicine(TCM) preparations containing S. miltiorrhiza, reduce the extraction cost, and relieve the pressure of clinical medication. The biosynthetic pathway of tanshinones contains multiple P450 enzymes, and the catalytic element with high efficiency is the basis of microbial production of tanshinones. In this study, the protein modification of CYP76AK1, a key P450-C20 hydroxylase in tanshinone pathway, was researched. The protein modeling methods SWISS-MODEL, Robetta, and AlphaFold2 were used, and the protein model was analyzed to obtain the reliable protein structure. The semi-rational design of mutant protein was carried out by molecular docking and homologous alignment. The key amino acid sites affecting the oxidation activity of CYP76AK1 were identified by molecular docking. The function of the obtained mutations was studied with yeast expression system, and the CYP76AK1 mutations with continuous oxidation function to 11-hydroxysugiol were obtained. Four key amino acid sites that affected the oxidation acti-vity were analyzed, and the reliability of three protein modeling methods was analyzed according to the mutation results. The effective protein modification sites of CYP76AK1 were reported for the first time in this study, which provides a catalytic element for different oxidation activities at C20 site for the study of the synthetic biology of tanshinones and lays a foundation for the analysis of the conti-nuous oxidation mechanism of P450-C20 modification.

[Effects of Cas9 expression on cell growth and production of natural products in Saccharomyces cerevisiae and optimization of CRISPR-Cas9 editing system].

CRISPR-Cas9 gene editing technology has been widely used in Saccharomyces cerevisiae.However, the effects of Cas9, as an exogenous protein, on the growth and production of natural products in S.cerevisiae are still unclear.In this study, Cas9 gene was expressed in S.cerevisiae by integration into the genome and construction into vectors, and two natural products, carotenoid and miltiradiene, were selected as the target products to study the effects of Cas9 expression on yeast growth and production capacity.The results showed that whether Cas9 was integrated into the genome or expressed by vectors, Cas9 inhibited the growth of S.cerevisiae, which was more obvious in the form of genome integration.When Cas9 was integrated into the genome, it had no effect on the production of carotenoid and miltiradiene by S.cerevisiae, but when Cas9 was expressed by vectors, the ability of S.cerevisiae to produce carotenoids and miltiradiene was significantly reduced.Therefore, in order to further efficiently knock out Cas9 after gene editing and minimize the adverse impact of Ura3 and Trp1 vectors, this study systematically explored the removal efficiency of the two vectors, and a plasmid capable of efficient gene editing was constructed, which optimized the application of CRISPR-Cas9 gene editing system in S.cerevisiae, and provided reference for the application of gene editing technology based on Cas9.

[Advances on the microbial synthesis of plant-derived diterpenoids].

Natural plant-derived diterpenoids are a class of compounds with diverse structures and functions. These compounds are widely used in pharmaceuticals, cosmetics and food additives industries because of their pharmacological properties such as anticancer, anti-inflammatory and antibacterial activities. In recent years, with the gradual discovery of functional genes in the biosynthetic pathway of plant-derived diterpenoids and the development of synthetic biotechnology, great efforts have been made to construct a variety of diterpenoid microbial cell factories through metabolic engineering and synthetic biology, resulting in gram-level production of many compounds. This article summarizes the construction of plant-derived diterpenoid microbial cell factories through synthetic biotechnology, followed by introducing the metabolic engineering strategies applied to improve plant-derived diterpenoids production, with the aim to provide a reference for the construction of high-yield plant-derived diterpenoid microbial cell factories and the industrial production of diterpenoids.

Functional characterization of CYP81C16 involved in the tanshinone biosynthetic pathway in Salvia miltiorrhiza.

Danshen, the dried roots and rhizomes of Salvia miltiorrhiza Bunge (S. miltiorrhiza), is widely used in the treatment of cardiovascular and cerebrovascular diseases. Tanshinones, the bioactive compounds from Danshen, exhibit a wide spectrum of pharmacological properties, suggesting their potential for future therapeutic applications. Tanshinone biosynthesis is a complex process involving at least six P450 enzymes that have been identified and characterized, most of which belong to the CYP76 and CYP71 families. In this study, CYP81C16, a member of the CYP71 clan, was identified in S. miltiorrhiza. An in vitro assay revealed that it could catalyze the hydroxylation of four para-quinone-type tanshinones, namely neocryptotanshinone, deoxyneocryptotanshinone, and danshenxinkuns A and B. SmCYP81C16 emerged as a potential broad-spectrum oxidase targeting the C-18 position of para-quinone-type tanshinones with an impressive relative conversion rate exceeding 90%. Kinetic evaluations andin vivo assays underscored its highest affinity towards neocryptotanshinone among the tested substrates. The overexpression of SmCYP81C16 promoted the accumulation of (iso)tanshinone in hairy root lines. The characterization of SmCYP81C16 in this study accentuates its potential as a pivotal tool in the biotechnological production of tanshinones, either through microbial or plant metabolic engineering.

[Research methods and progress on the reduction effects of vehicle emission pollutants by street canyon greening.] / è¡—è°·ç»¿åŒ–æ¶ˆå‡æœºåŠ¨è½¦æŽ’æ”¾æ±¡æŸ“ç‰©çš„ç ”ç©¶æ–¹æ³•ä¸Žè¿›å±•.

With the development of high-density and high-rise buildings on both sides of the street, widespread attention has been paid to the applicability of the traditional greening model of 'the more trees, the better atmospheric environment' in dealing with air pollution in urban street canyons. Clarifying the characteristics of street canyons greening and its planting design pattern on the reduction of emission pollutants by vehicles is an important prerequisite for the improvement of air quality in the street canyons. Based on literature review, we compared the applicability and limitations of the three methods, including field observation, wind tunnel test, and numerical simulation. We further analyzed the effects of roadside trees and hedges on the dispersion and deposition of air pollutants, and put forward a framework of adaptive greening design for air quality improvement. Finally, we proposed that future studies should address the creation of graphic languages for roadside greening design, the development of technical guidelines for evaluating the exposure of air pollution, and the optimization of parameterization schemes for the physical processes of greening effect in computational fluid dynamics models. Overall, our review could provide ideas and reference for the subsequent research.

[Planning approach of urban blue-green space based on local climate optimization: A review]. / åŸºäºŽå±€åœ°æ°”å€™ä¼˜åŒ–çš„åŸŽå¸‚è“ç»¿ç©ºé—´è§„åˆ’é€”å¾„ç ”ç©¶è¿›å±•.

Local climatic problems are widespread in cities under high-density and high-intensity construction mode in China, including heat island effect, poor ventilation, and frequent haze wea-ther. Urban blue-green space is vital for mitigating wind and heat, and for improving air quality, and therefore has become a hotspot of urban planning and design research dealing with climatic problems. Here, we reviewed the climate effects of urban blue-green space. In particular, we summarized the research progress of planning approaches to cool island landscape features optimization, cooling island configuration, ventilation corridor network connection and ventilation corridor interface control based on the levels of planning layout and network construction. We proposed a basic framework of urban blue-green space planning optimizing local climate by combining intelligent simulation platform, evaluation index system, planning and design guide, and guarantee mechanism for implement, aiming to provide key evidence for urban climate adaption and design.